【作者】 许端敏；

【导师】 李玉光；

【作者基本信息】 汕头大学 ， 心血管内科， 2003， 硕士

【摘要】 背景与目的：经皮腔内成形术和支架术是目前治疗动脉狭窄性疾病的重要手段，然而术后再狭窄发生率高，影响其远期疗效。术后的主要病理改变是血管平滑肌细胞(VSMC)激活、迁移、增殖，新生内膜形成。细胞外基质和基底膜的降解是VSMC迁移的前提条件，基质金属蛋白酶3(MMP3)表达异常是导致细胞外基质降解的重要原因。本课题在建立大鼠主动脉球囊损伤模型的基础上，模拟再狭窄的病理变化，应用雷帕霉素进行干预，研究内膜损伤后雷帕霉素对内膜增殖的影响，以及MMP3的变化，探讨雷帕霉素预防再狭窄的可能机制。 材料与方法：健康成年雄性SD大鼠150只，随机分为4组：第1组为假手术组(n=20)；第2组为单纯球囊损伤(球囊损伤组，n=40)；第3组为球囊损伤+羧甲基纤维素钠干预(羧甲基纤维素钠组，n=40)；第4组为球囊损伤+雷帕霉素干预(雷帕霉素组，n=40)。术后3天、7天、14天、21天、28天处死动物。病理切片分析血管内膜面积、内膜平均厚度、中膜面积，计算内膜／中膜面积比，计数术后3天内膜有核细胞数。免疫组织化学high-SABC法分别检测MMP3、 汕头大学医学院硕士研究生毕业论文PCNA、Q一Actin在腹主动脉上的表达。电镜观察VSMC表型的变化。测定血脂、肾功能和外周血白细胞。测量实验前后动物的体重。结果:(l)雷帕霉素组新生内膜面积比梭甲基纤维素钠组减少36.4%(0.14士0.03二2 vs 0.23士0.O6nunZ at14 days，p(0.01)，内膜/中膜比减少28.9%(17.7士3.37%vs 28.86士10.25%at 14days，p<0.01)。(2)术后3天雷帕霉素组内膜有核细胞数比梭甲基纤维素钠组明显减少(8.88士3.04 vs12.87士3.44，p<0.05)。(3)球囊损伤后3天动脉壁MMP3表达逐渐增加，以14天最明显，以后逐渐下降，雷帕霉素组MMP3的表达明显减少。(4)损伤早期MMP3的表达与新生内膜面积、内膜厚度成正相关，r分别为0.821和0.771，p均<0.05;(5)电镜表现:假手术组vSMC呈收缩表型，肌丝多，细胞器少;球囊损伤后VSMC呈合成表型，肌丝少，大量细胞器;雷帕霉素组VSMC表型介于收缩表型与合成表型之间。雷帕霉素组电镜下未见VSMC穿越弹力膜小窗现象。(6)雷帕霉素组血胆固醇升高，肾功能、外周血白细胞计数无改变;大剂量雷帕霉素组出现间质性肺炎。结论:(l)动脉损伤后血管壁MMP3的表达呈动态变化，3天后表达开始增加，14天达高峰，以后逐渐下降;本实验首次证明雷帕霉素可以减少术后MMP3的表达。(2)雷帕霉素减少内膜有核细胞数，减少内膜面积和内膜/中膜比，说明雷帕霉素可以抑制大鼠腹主动脉球囊损伤术后体内VSMC的迁移，减轻术后新生内膜增生程度。(3)雷帕霉素可以影响VSMC表型的改变。(4)小剂量的雷帕霉素对造血系汕头大学医学院硕士研究生毕业论文统、肾功能影响不大;大剂量的雷帕霉素可能引起间质性肺炎。更多还原

【Abstract】 Background and objective: Although percutaneous transluminal angioplasty and stent implantation are highly procedures to reduce the severity of stenotic artery disease, their long-term success are significantly limited by high rate of restenosis. Several mechanisms have been implicated in the development of restenosis post-injury, including vascular smooth muscle cell(VSMC) activation, migration and proliferation. Extracellular matrix and basement membranes degradation are premising Condition for VSMC migration. Matrix metalloproteinase3 (MMP3) play an important role in the degradation of extracellular matrix. In the present study, we investigated the change of MMP3 in a rat model of restenosis post angioplasty and whether rapamycin administration could reduce neointimal thickening and the mechanism by which rapamycin inhibits VSMCs in vivo.Methods: One hundred and fifty healthy adult male rat were randomized divided into four groups: (1) Sham operation group(n=20). (2) Balloon group (n=40) :received balloon injury only.(3) Cellulose group(n=40) :received the vehicle solution. (4) Rapamycin group(n=40):intramusclar administration of rapamycin (suspended in solution containing 0.2% sodium carboxymethyl cellulose) started 3 days before injury at a dose of 0. 5 mg/kg and continued for 14 days at a dose of 0. 25 mg/kg. Animals were harvested at 3 days,7 days, 14 days, 21 days and 28 days after injury. Abdominal aortas were analyzed for intimal area, mean intimal thickness and intimal/media area ratio. Intimal nuclear cells were counted at 3 days after injury. MMP3 and PCNA and a -Actin within the vessel wall were determined by immmunohischemistry stain. VSMC phenotype was observed by electron microscope. Blood lipid, renal function, white blood cells and body weight were determined. Results: (1) Neointimal area and intimal/media area ratio 14 days after injury showed a 36.4% and 28.9% reduced in the animals receiving rapamycin compared with those receiving cellulose respectively(0. 14 ± 0. 03 mm2 vs 0.23 ± 0. 06mm2 and 17. 70 ± 3. 37% vs 28. 86 ± 10. 25%, respectively, P<0. 01). (2) Nuclear cell counts were lower in intima of rapamycin-treated arteries than in cellulose arteries (8. 88 ± 3. 04 vs 12. 87 ± 3. 44 at 3 days, p<0. 05). (3)MMP3 in vessel wall increased at 3 daysand reached a peak at 14 days after injury. The expression of MMP3 was lower in rapamycin group. (4)MMP3 related to intimal area and mean intimal thickness (r=0.821 and 0.771, respectively). (5) There were a great number of mitochondrion and little myofilament in VSMC 7 days after injury. Rapamycin administration decreased organelles and increased myof ilament. No VSMC of media passed through micro-window in internal elastic lamina into intima in rapamycin group. (6)Blood cholesterol but not renal fuction and white blood cell was higher in rapamycin group. Interstitial pneumonia occurred in the animals receiving high- dose rapamycin. Conclusions:(1) The expression of MMP3 in aorta wall after injury is a developing procedure. Our study first demonstrates rapamycin can result in MMP3 decrease. (2)Rapamycin can reduce the degree of neointimal hyperplasia via inhibiting VSMC migration in vivo. (3)The phenotype of VSMC can be changed by rapamycin. (4)Low-dose rapamycin has little effect on Hematopoietic system and renal fuction. High-dose rapamycin can cause interstitial pneumonia.更多还原